Checkpoint inhibitor and a whole cell mycobacterium for use in cancer therapy

ABSTRACT

An immunomodulator for use in the treatment, reduction, inhibition or control of a neoplastic disease in a patient intended to undergo checkpoint inhibition therapy, simultaneously, separately or sequentially with administration of the immunomodulator. The immunomodulator preferably comprises a whole cell  Mycobacterium , for example,  M. vaccae  or  M. obuense.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/112,430, filed Aug. 24, 2018, which is a continuation of U.S.application Ser. No. 15/104,890, filed Jun. 15, 2016, which is aNational Stage Application of PCT/GB2014/053717, filed Dec. 16, 2014,which claims benefit of Application No. 1322725.1, filed Dec. 20, 2013in United Kingdom and which applications are incorporated herein byreference. To the extent appropriate, a claim of priority is made toeach of the above disclosed applications.

FIELD OF THE INVENTION

The present invention relates to the field of cancer therapy. Inparticular, the present invention relates to a method of preventing,treating or inhibiting the development of tumours and/or metastases in asubject.

BACKGROUND OF THE INVENTION

In humans with advance cancer, anti-tumour immunity is often ineffectivedue to the tightly regulated interplay of pro- and anti-inflammatory,immune-stimulatory and immunosuppressive signals. For example, loss ofthe anti-inflammatory signals leads to chronic inflammation andprolonged proliferative signalling. Interestingly, cytokines that bothpromote and suppress proliferation of the tumour cells are produced atthe tumour site. It is the imbalance between the effects of thesevarious processes that results in tumour promotion.

To date, a major barrier to attempts to develop effective immunotherapyfor cancer has been an inability to break immunosuppression at thecancer site and restore normal networks of immune reactivity. Thephysiological approach of immunotherapy is to normalize the immunereactivity so that, for example, the endogenous tumour antigens would berecognized and effective cytolytic responses would be developed againsttumour cells. Although it was once unclear if tumour immunosurveillanceexisted, it is now believed that the immune system constantly monitorsand eliminates newly transformed cells. Accordingly, cancer cells mayalter their phenotype in response to immune pressure in order to escapeattack (immunoediting) and upregulate expression of inhibitory signals.Through immunoediting and other subversive processes, primary tumour andmetastasis maintain their own survival.

One of the major mechanisms of anti-tumour immunity subversion is knownas ‘T-cell exhaustion’, which results from chronic exposure to antigensand is characterized by the up-regulation of inhibitory receptors. Theseinhibitory receptors serve as immune checkpoints in order to preventuncontrolled immune reactions.

PD-1 and co-inhibitory receptors such as cytotoxic T-lymphocyte antigen4 (CTLA-4, B and T Lymphocyte Attenuator (BTLA; CD272), T cellimmunoglobulin and Mucin domain-3 (Tim-3), Lymphocyte Activation Gene-3(Lag-3; CD223), and others are often referred to as a checkpointregulators. They act as molecular “tollbooths,” which allowextracellular information to dictate whether cell cycle progression andother intracellular signaling processes should proceed.

In addition to specific antigen recognition through the TCR, T-cellactivation is regulated through a balance of positive and negativesignals provided by co-stimulatory receptors. These surface proteins aretypically members of either the TNF receptor or B7 superfamilies.Agonistic antibodies directed against activating co-stimulatorymolecules and blocking antibodies against negative co-stimulatorymolecules may enhance T-cell stimulation to promote tumour destruction.

Programmed Cell Death Protein 1, (PD-1 or CD279), a 55-kD type 1transmembrane protein, is a member of the CD28 family of T cellco-stimulatory receptors that include immunoglobulin superfamily memberCD28, CTLA-4, inducible co-stimulator (ICOS), and BTLA. PD-1 is highlyexpressed on activated T cells and B cells. PD-1 expression can also bedetected on memory T-cell subsets with variable levels of expression.Two ligands specific for PD-1 have been identified: programmeddeath-ligand 1 (PD-L1, also known as B7-H1 or CD274) and PD-L2 (alsoknown as B7-DC or CD273). PD-L1 and PD-L2 have been shown todown-regulate T cell activation upon binding to PD-1 in both mouse andhuman systems (Okazaki et al., Int Immunol., 2007; 19: 813-824). Theinteraction of PD-1 with its ligands, PD-L1 and PD-L2, which areexpressed on antigen-presenting cells (APCs) and dendritic cells (DCs),transmits negative regulatory stimuli to down-modulate the activated Tcell immune response. Blockade of PD-1 suppresses this negative signaland amplifies T cell responses.

Numerous studies indicate that the cancer microenvironment manipulatesthe PD-L1-/PD-1 signalling pathway and that induction of PD-L1expression is associated with inhibition of immune responses againstcancer, thus permitting cancer progression and metastasis. ThePD-L1/PD-1 signalling pathway is a primary mechanism of cancer immuneevasion for several reasons. First, and most importantly, this pathwayis involved in negative regulation of immune responses of activated Teffector cells, found in the periphery. Second, PD-L1 is up-regulated incancer microenvironments, while PD-1 is also up-regulated on activatedtumour infiltrating T cells, thus possibly potentiating a vicious cycleof inhibition. Third, this pathway is intricately involved in bothinnate and adaptive immune regulation through bi-directional signalling.These factors make the PD-1/PD-L1 complex a central point through whichcancer can manipulate immune responses and promote its own progression.

The first immune-checkpoint inhibitor to be tested in a clinical trialwas ipilimumab (Yervoy, Bristol-Myers Squibb), an CTLA-4 mAb. CTLA-4belongs to the immunoglobulin superfamily of receptors, which alsoincludes PD-1, BTLA, TIM-3, and V-domain immunoglobulin suppressor of Tcell activation (VISTA).

Anti-CTLA-4 mAb is a powerful checkpoint inhibitor which removes “thebreak” from both naïve and antigen-experienced cells. Therapy enhancesthe antitumor function of CD8+ T cells, increases the ratio of CD8+ Tcells to Foxp3+ T regulatory cells, and inhibits the suppressivefunction of T regulatory cells. The major drawback to anti-CTLA-4 mAbtherapy is the generation of autoimmune toxicities due to on-targeteffects of an over-exuberant immune system which has lost the ability toturn itself down. It has been reported that up to 25% of patientstreated with ipilimumab developed serious grade 3-4 adverseevents/autoimmune-type side effects including dermatitis, enterocolitis,hepatitis, endocrinopathies (including hypophysitis, thyroiditis, andadrenalitis), arthritis, uveitis, nephritis, and aseptic meningitis. Incontrast to the anti-CTLA-4 experience, anti-PD-1 therapy appears to bebetter-tolerated and induces a relatively lower rate of autoimmune-typeside effects.

TIM-3 has been identified as another important inhibitory receptorexpressed by exhausted CD8+ T cells. In mouse models of cancer, it hasbeen shown that the most dysfunctional tumour-infiltrating CD8+ T cellsactually co-express PD-1 and TIM-3.

LAG-3 is another recently identified inhibitory receptor that acts tolimit effector T-cell function and augment the suppressive activity of Tregulatory cells. It has recently been revealed that PD-1 and LAG-3 areextensively co-expressed by tumour-infiltrating T cells in mice, andthat combined blockade of PD-1 and LAG-3 provokes potent synergisticantitumor immune responses in mouse models of cancer.

PD-1 pathway blockade can be combined with vaccines or otherimmunomodulatory antibodies for improved therapeutic efficacy (Hirano,F. et al, Cancer Res., 65(3): 1089-1096 (2005); Li, B. et al, Clin.Cancer Res., 15: 1507-1509 (2009); and Curran, M. A. et al, Proc. Natl.Acad. Set, 107(9):4275-4280 (2010)).

Currently, antagonist mAbs against both PD-1 and their ligand PD-L1 arein various stages of development for the treatment of cancer, and recenthuman trials have shown promising results in cancer patients withadvanced, treatment-refractory disease.

The first of the agents blocking the B7-H1/PD-1 pathway to enter phase Iclinical trials was Nivolumab (MDX-1106/BMS-936558/ONO-4538), a fullyhuman IgG4 anti-PD-1 mAb developed by Bristol-Myers Squibb. Another PD-1mAb undergoing clinical evaluation is CT-011, a humanized IgG1 mAbspecific for PD-1 developed by CureTech Ltd. Other agents includeLambrolizumab (MK-3475—Merck), a humanized monoclonal IgG4 PD-1antibody; BMS-936559, a fully human IgG4 PD-L1 antibody and Roche'sMPDL3280A, a human monoclonal antibody that targets the PD-L1 pathway.

Accordingly, an aim of the present invention is a combination therapyfor treating cancer comprising an immunomodulator and blockade ofcheckpoint inhibitors with the potential to elicit potent and durableimmune responses.

SUMMARY OF THE INVENTION

The present invention provides an effective method for treating and/orpreventing cancer and/or the establishment of metastases byadministering a checkpoint inhibitor which acts synergistically with awhole cell Mycobacterium.

In a first aspect of the invention, there is an immunomodulator for usein the treatment, reduction, inhibition or control of a neoplasticdisease in a patient intended to undergo checkpoint inhibition therapysimultaneously, separately or sequentially with administration of theimmunomodulator.

In a second aspect of the invention, there is a method of treating,reducing, inhibiting or controlling a neoplasia, tumour or cancer in asubject, wherein said method comprises simultaneously, separately orsequentially administering to the subject, (i) a checkpoint inhibitor,and (ii) an immunomodulator, wherein said method results in enhancedtherapeutic efficacy relative to administration of the checkpointinhibitor or immunomodulator alone.

In a third aspect of the invention, there is a method of treating,reducing, inhibiting or controlling a neoplasia, tumour or cancer in asubject, wherein said method comprises simultaneously, separately orsequentially administering to the subject, (i) a sub-therapeutic amountand/or duration of checkpoint inhibitor, and (ii) an immunomodulator,wherein said method results in enhanced therapeutic efficacy relative toadministration of the checkpoint inhibitor or immunomodulator alone.

The present invention therefore provides a combination therapy ofcheckpoint inhibitor therapy together with a specific type ofimmunotherapy comprising administration of an immunomodulator. Theinventors have found that the combination of both therapies issynergistic beyond simple additive effects of each therapy usedindividually.

DESCRIPTION OF THE DRAWINGS

The invention is described with reference to the following drawings, inwhich:

FIG. 1 shows the effect of a preparation of heat-killed Mycobacteriumobuense (NCTC 13365) with or without co-administration of a checkpointinhibitor (ant-PD-L1 mAb), in a xenograft model of pancreatic cancer(KPC cells injected subcutaneously).

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a method for treating, reducing, inhibiting orcontrolling a neoplasia, tumour or cancer in a subject involvingadministering an immunomodulator and a checkpoint inhibitor. It is basedupon the discovery that administration of an immunomodulator (whole cellheat-killed Mycobacterium) in combination with an anti-PD-L1 antibody (acheckpoint inhibitor) results in synergistic anti-tumour activity and/orantitumor activity that is more potent than administration ofimmunomodulator or anti-PD-L1 antibody alone.

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

A “checkpoint inhibitor” is an agent which acts on surface proteinswhich are members of either the TNF receptor or B7 superfamilies,including agents which bind to negative co-stimulatory moleculesselected from CTLA-4, PD-1, TIM-3, BTLA, VISTA, LAG-3, and/or theirrespective ligands, including PD-L1. (Mellman et al., supra).

An immunomodulator, as defined according to the present invention, is acomponent which stimulates innate and type-1 immunity, including Th1 andmacrophage activation and cytotoxic cell activity, as well asindependently down-regulating inappropriate anti-Th2 responses viaimmunoregulatory mechanisms.

The terms “tumour,” “cancer” and “neoplasia” are used interchangeablyand refer to a cell or population of cells whose growth, proliferationor survival is greater than growth, proliferation or survival of anormal counterpart cell, e.g. a cell proliferative or differentiativedisorder. Typically, the growth is uncontrolled. The term “malignancy”refers to invasion of nearby tissue. The term “metastasis” refers tospread or dissemination of a tumour, cancer or neoplasia to other sites,locations or regions within the subject, in which the sites, locationsor regions are distinct from the primary tumour or cancer.

The terms “Programmed Death 1,” “Programmed Cell Death 1,” “ProteinPD-1,” “PD-1,” and “PD1,” are used interchangeably, and includevariants, isoforms, species homologs of human PD-1, and analogs havingat least one common epitope with PD-1. The complete PD-1 sequence can befound under GenBank Accession No. U64863.

The terms “cytotoxic T lymphocyte-associated antigen-4,” “CTLA-4,”“CTLA4,” and “CTLA-4 antigen” (see, e.g., Murata, Am. J. Pathol. (1999)155:453-460) are used interchangeably, and include variants, isoforms,species homologs of human CTLA-4, and analogs having at least one commonepitope with CTLA-4 (see, e.g., Balzano (1992) Int. J. Cancer Suppl.7:28-32). The complete CTLA-4 nucleic acid sequence can be found underGenBank Accession No. L15006.

As used herein, “sub-therapeutic dose” means a dose of a therapeuticcompound (e.g., an antibody) or duration of therapy which is lower thanthe usual or typical dose of the therapeutic compound or therapy ofshorter duration, when administered alone for the treatment of cancer.For example, a sub-therapeutic dose of CTLA-4 antibody is a single doseof the antibody at less than about 3 mg/kg, i.e., the known dose ofanti-CTLA-4 antibody.

The term “therapeutically effective amount” is defined as an amount of acheckpoint inhibitor, in combination with an immunomodulator, thatpreferably results in a decrease in severity of disease symptoms, anincrease in frequency and duration of disease symptom-free periods, or aprevention of impairment or disability due to the disease affliction.The terms “effective amount” or “pharmaceutically effective amount”refer to a sufficient amount of an agent to provide the desiredbiological or therapeutic result. That result can be reduction,amelioration, palliation, lessening, delaying, and/or alleviation of oneor more of the signs, symptoms, or causes of a disease, or any otherdesired alteration of a biological system. In reference to cancer, aneffective amount may comprise an amount sufficient to cause a tumour toshrink and/or to decrease the growth rate of the tumour (such as tosuppress tumour growth) or to prevent or delay other unwanted cellproliferation. In some embodiments, an effective amount is an amountsufficient to delay development, or prolong survival or inducestabilisation of the cancer or tumour.

In some embodiments, a therapeutically effective amount is an amountsufficient to prevent or delay recurrence. A therapeutically effectiveamount can be administered in one or more administrations. Thetherapeutically effective amount of the drug or combination may resultin one or more of the following: (i) reduce the number of cancer cells;(ii) reduce tumour size; (iii) inhibit, retard, slow to some extent andpreferably stop cancer cell infiltration into peripheral organs; (iv)inhibit (i.e., slow to some extent and preferably stop) tumourmetastasis; (v) inhibit tumour growth; (vi) prevent or delay occurrenceand/or recurrence of tumour; and/or (vii) relieve to some extent one ormore of the symptoms associated with the cancer.

For example, for the treatment of tumours, a “therapeutically effectivedosage” may induce tumour shrinkage by at least about 5% relative tobaseline measurement, such as at least about 10%, or about 20%, or about60% or more. The baseline measurement may be derived from untreatedsubjects.

A therapeutically effective amount of a therapeutic compound candecrease tumour size, or otherwise ameliorate symptoms in a subject. Oneof ordinary skill in the art would be able to determine such amountsbased on such factors as the subject's size, the severity of thesubject's symptoms, and the particular 10 composition or route ofadministration selected.

The term “immune response” refers to the action of, for example,lymphocytes, antigen presenting cells, phagocytic cells, granulocytes,and soluble macromolecules produced by the above cells or the liver(including antibodies, cytokines, and complement) that results inselective damage to, destruction of, or elimination from the human bodyof cancerous cells.

The term “antibody” as referred to herein includes whole antibodies andany antigen-binding fragment (i.e., “antigen-binding portion”) or singlechains thereof.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to a receptor andits ligand (e.g., PD-1). including: (i) a Fab fragment, (ii) a F(ab′) 2fragment; (iii) a Fd fragment consisting of the VH and CHI domains; (iv)a Fv fragment, (v) a dAb fragment (Ward et al, Nature, 341:544-546(1989)), which consists of a VH domain; and (vi) an isolatedcomplementarity determining region (CDR). Single chain antibodies arealso intended to be encompassed within the term “antigen-bindingportion” of an antibody. These antibody fragments are obtained usingconventional techniques known to those with skill in the art, and thefragments are screened for utility in the same manner as are intactantibodies.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.

The term “human antibody”, as used herein, is intended to includeantibodies having variable regions in which both the framework and CDRregions are derived from human germline immunoglobulin sequences.

The term “humanized antibody” is intended to refer to antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences. Additional framework region modifications may be made withinthe human framework sequences.

In addition to antibodies, other biological molecules may act ascheckpoint inhibitors, including peptides having binding affinity to theappropriate target.

The term “treatment” or “therapy” refers to administering an activeagent with the purpose to cure, heal, alleviate, relieve, alter, remedy,ameliorate, improve, or affect a condition (e.g., a disease), thesymptoms of the condition, or to prevent or delay the onset of thesymptoms, complications, biochemical indicia of a disease, or otherwisearrest or inhibit further development of the disease, condition, ordisorder in a statistically significant manner.

As used herein, the term “subject” is intended to include human andnon-human animals. Preferred subjects include human patients in need ofenhancement of an immune response. The methods are particularly suitablefor treating human patients having a disorder that can be treated byaugmenting the T-cell mediated immune response. In a particularembodiment, the methods are particularly suitable for treatment ofcancer cells in vivo.

As used herein, the terms “concurrent administration” or “concurrently”or “simultaneous” mean that administration occurs on the same day. Theterms “sequential administration” or “sequentially” or “separate” meanthat administration occurs on different days.

“Simultaneous” administration, as defined herein, includes theadministration of the immunomodulator and agent or procedure comprisingcheckpoint inhibitor therapy within about 2 hours or about 1 hour orless of each other, even more preferably at the same time.

“Separate” administration, as defined herein, includes theadministration of the immunomodulator and agent or procedure comprisingcheckpoint inhibitor therapy, more than about 12 hours, or about 8hours, or about 6 hours or about 4 hours or about 2 hours apart.

“Sequential” administration, as defined herein, includes theadministration of the immunomodulator and agent or procedure comprisingcheckpoint inhibitor therapy each in multiple aliquots and/or dosesand/or on separate occasions. The immunomodulator may be administered tothe patient after before and/or after administration of the checkpointinhibitor. Alternatively, the immunomodulator is continued to be appliedto the patient after treatment with a checkpoint inhibitor.

The use of the alternative (e.g., “or”) should be understood to meaneither one, both, or any combination thereof of the alternatives. Asused herein, the indefinite articles “a” or “an” should be understood torefer to “one or more” of any recited or enumerated component.

As used herein, “about” means within an acceptable error range for theparticular value as determined by one of ordinary skill in the art,which will depend in part on how the value is measured or determined,i.e., the limitations of the measurement system. For example, “about”can mean within 1 or more than 1 standard deviation per the practice inthe art. Alternatively, “about” can mean a range of up to 20%. Whenparticular values are provided in the application and claims, unlessotherwise stated, the meaning of “about” should be assumed to be withinan acceptable error range for that particular value.

In one aspect of the present invention the immunomodulator comprisesheat-killed Mycobacterium, preferably a whole cell Mycobacterium.Examples of mycobacterial species for use in the present inventioninclude M. vaccae, M. thermoresistibile, M. flavescens, M. duvalii, M.phlei, M. obuense, M. parafortuitum, M. sphagni, M. aichiense, M.rhodesiae, M. neoaurum, M. chubuense, M. tokaiense, M. komossense, M.aurum, M. w, M. tuberculosis, M. microti; M. africanum; M. kansasii, M.marinum; M. simiae; M. gastri; M. nonchromogenicum; M. terrae; M.triviale; M. gordonae; M. scrofulaceum; M. paraffinicum; M.intracellulare; M. avium; M. xenopi; M. ulcerans; M. diernhoferi, M.smegmatis; M. thamnopheos; M. flavescens; M. fortuitum; M. peregrinum;M. chelonei; M. paratuberculosis; M. leprae; M. lepraemurium andcombinations thereof.

Preferably, the heat-killed Mycobacterium is non-pathogenic. Thenon-pathogenic heat-killed Mycobacterium is preferably selected from M.vaccae, M. obuense, M. parafortuitum, M. aurum, M. indicus pranii, M.phlei and combinations thereof. More preferably the non-pathogenicheat-killed Mycobacterium is a rough variant. The amount ofMycobacterium administered to the patient is sufficient to elicit aprotective immune response in the patient such that the patient's immunesystem is able to mount an effective immune response to the cancer ortumour. In certain embodiments of the invention, there is provided acontainment means comprising the effective amount of heat-killedMycobacterium for use in the present invention, which typically may befrom 10³ to 10¹¹ organisms, preferably from 10⁴ to 10¹⁰ organisms, morepreferably from 10⁶ to 10¹⁰ organisms, and even more preferably from 10⁶to 10⁹ organisms. The effective amount of heat-killed Mycobacterium foruse in the present invention may be from 10³ to 10¹¹ organisms,preferably from 10⁴ to 10¹⁰ organisms, more preferably from 10⁶ to 10¹⁰organisms, and even more preferably from 10⁶ to 10⁹ organisms. Mostpreferably the amount of heat-killed Mycobacterium for use in thepresent invention is from 10⁷ to 10⁹ cells or organisms. Typically, thecomposition according to the present invention may be administered at adose of from 10⁸ to 10⁹ cells for human and animal use. Alternativelythe dose is from 0.01 mg to 1 mg or 0.1 mg to 1 mg organisms presentedas either a suspension or dry preparation.

M. vaccae and M. obuense are particularly preferred.

M. vaccae and M. obuense induce a complex immune response in the host.Treatment with these preparations will stimulate innate and type-1immunity, including Th1 and macrophage activation and cytotoxic cellactivity. They also independently down-regulate inappropriate Th2responses via immunoregulatory mechanisms. This restores the healthybalance of the immune system.

The present invention may be used to treat a neoplastic disease, such assolid or non-solid cancers. As used herein, “treatment” encompasses theprevention, reduction, control and/or inhibition of a neoplasticdisease. Such diseases include a sarcoma, carcinoma, adenocarcinoma,melanoma, myeloma, blastoma, glioma, lymphoma or leukemia. Exemplarycancers include, for example, carcinoma, sarcoma, adenocarcinoma,melanoma, neural (blastoma, glioma), mesothelioma andreticuloendothelial, lymphatic or haematopoietic neoplastic disorders(e.g., myeloma, lymphoma or leukemia). In particular aspects, aneoplasm, tumour or cancer includes a lung adenocarcinoma, lungcarcinoma, diffuse or interstitial gastric carcinoma, colonadenocarcinoma, prostate adenocarcinoma, esophagus carcinoma, breastcarcinoma, pancreas adenocarcinoma, ovarian adenocarcinoma,adenocarcinoma of the adrenal gland, adenocarcinoma of the endometriumor uterine adenocarcinoma.

Neoplasia, tumours and cancers include benign, malignant, metastatic andnon-metastatic types, and include any stage (I, II, III, IV or V) orgrade (G1, G2, G3, etc.) of neoplasia, tumour, or cancer, or aneoplasia, tumour, cancer or metastasis that is progressing, worsening,stabilized or in remission. Cancers that may be treated according to theinvention include but are not limited to cells or neoplasms of thebladder, blood, bone, bone marrow, brain, breast, colon, esophagus,gastrointestines, gum, head, kidney, liver, lung, nasopharynx, neck,ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition,the cancer may specifically be of the following histological type,though it is not limited to the following: neoplasm, malignant;carcinoma; carcinoma, undifferentiated; giant and spindle cellcarcinoma; small cell carcinoma; papillary carcinoma; squamous cellcarcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrixcarcinoma; transitional cell carcinoma; papillary transitional cellcarcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma;hepatocellular carcinoma; combined hepatocellular carcinoma andcholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma;adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposiscoli; solid carcinoma; carcinoid tumour, malignant; bronchiolo-alveolaradenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma;acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clearcell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma;papillary and follicular adenocarcinoma; nonencapsulating sclerosingcarcinoma; adrenal cortical carcinoma; endometroid carcinoma; skinappendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma;ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma;papillary cystadenocarcinoma; papillary serous cystadenocarcinoma;mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cellcarcinoma; infiltrating duct carcinoma; medullary carcinoma; lobularcarcinoma; inflammatory carcinoma; Paget's disease, mammary; acinar cellcarcinoma; adenosquamous carcinoma; adenocarcinoma with squamousmetaplasia; thymoma, malignant; ovarian stromal tumour, malignant;thecoma, malignant; granulosa cell tumour, malignant; androblastoma,malignant; Sertoli cell carcinoma; Leydig cell tumour, malignant; lipidcell tumour, malignant; paraganglioma, malignant; extra-mammaryparaganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignantmelanoma; amelanotic melanoma; superficial spreading melanoma; malignantmelanoma in giant pigmented nevus; epithelioid cell melanoma; bluenevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma,malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma;embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma;mixed tumour; Mullerian mixed tumour; nephroblastoma; hepatoblastoma;carcinosarcoma; mesenchymoma, malignant; Brenner tumour, malignant;phyllodes tumour, malignant; synovial sarcoma; mesothelioma, malignant;dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii,malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma;hemangioendothelioma, malignant; Kaposi's sarcoma; hemangiopericytoma,malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma;chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma;giant cell tumour of bone; Ewing's sarcoma; odontogenic tumour,malignant; ameloblastic odontosarcoma; ameloblastoma, malignant;ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma,malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillaryastrocytoma; astroblastoma; glioblastoma; oligodendroglioma;oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma;ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactoryneurogenic tumour; meningioma, malignant; neurofibrosarcoma;neurilemmoma, malignant; granular cell tumour, malignant; malignantlymphoma; Hodgkin's disease; Hodgkin's; paragranuloma; malignantlymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse;malignant lymphoma, follicular; mycosis fungoides; other specifiednon-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mastcell sarcoma; immunoproliferative small intestinal disease; leukemia;lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcomacell leukemia; myeloid leukemia; basophilic leukemia; eosinophilicleukemia; monocytic leukemia; mast cell leukemia; megakaryoblasticleukemia; myeloid sarcoma; and hairy cell leukemia. Preferably, theneoplastic disease may be tumours associated with a cancer selected fromprostate cancer, liver cancer, renal cancer, lung cancer, breast cancer,colorectal cancer, pancreatic cancer, brain cancer, hepatocellularcancer, lymphoma, leukaemia, gastric cancer, cervical cancer, ovariancancer, thyroid cancer, melanoma, head and neck cancer, skin cancer andsoft tissue sarcoma and/or other forms of carcinoma. The tumour may bemetastatic or a malignant tumour.

More preferably, the neoplastic disease to be treated is pancreaticcancer, breast cancer, lung cancer, prostate cancer and skin cancer.Most preferably, the neoplastic disease to be treated is pancreaticcancer.

In an embodiment of the invention, the checkpoint inhibitor therapy, incombination therapy with an immunomodulator, most preferably a wholecell, heat-killed Mycobacterium, is used to reduce or inhibit metastasisof a primary tumour or cancer to other sites, or the formation orestablishment of metastatic tumours or cancers at other sites distalfrom the primary tumour or cancer thereby inhibiting or reducing tumouror cancer relapse or tumour or cancer progression.

In a further embodiment of the invention, there is provided acombination therapy for treating cancer, comprising an immunomodulatorand blockade of checkpoint inhibitors with the potential to elicitpotent and durable immune responses with enhanced therapeutic benefitand more manageable toxicity.

In a further embodiment of the invention, there is provided acombination therapy for treating cancer, comprising an immunomodulatorwhich; (i) stimulates innate and type-1 immunity, including Th1 andmacrophage activation and cytotoxic cell activity, and, (ii)independently down-regulates inappropriate Th2 responses viaimmunoregulatory mechanisms; and, blockade of a checkpoint inhibitor,optionally wherein the immunomodulator is a whole cell Mycobacteriumselected from M. vaccae or M. obuense.

In an embodiment of the invention is provided a method for treatingcancer and/or preventing the establishment of metastases by employing acheckpoint inhibitor which act synergistically with a whole cellMycobacterium.

In further embodiments, methods of the invention include, one or more ofthe following: 1) reducing or inhibiting growth, proliferation, mobilityor invasiveness of tumour or cancer cells that potentially or do developmetastases, 2) reducing or inhibiting formation or establishment ofmetastases arising from a primary tumour or cancer to one or more othersites, locations or regions distinct from the primary tumour or cancer;3) reducing or inhibiting growth or proliferation of a metastasis at oneor more other sites, locations or regions distinct from the primarytumour or cancer after a metastasis has formed or has been established,4) reducing or inhibiting formation or establishment of additionalmetastasis after the metastasis has been formed or established, 5)prolonged overall survival, 6) prolonged progression free survival, or7) disease stabilisation.

In an embodiment of the invention, administration of the checkpointinhibitor therapy, in combination therapy with an immunomodulator,preferably a whole cell, heat-killed Mycobacterium, provides adetectable or measurable improvement in a condition of a given subject,such as alleviating or ameliorating one or more adverse (physical)symptoms or consequences associated with the presence of a cellproliferative or cellular hyperproliferative disorder, neoplasia, tumouror cancer, or metastasis, i e., a therapeutic benefit or a beneficialeffect.

A therapeutic benefit or beneficial effect is any objective orsubjective, transient, temporary, or long-term improvement in thecondition or pathology, or a reduction in onset, severity, duration orfrequency of an adverse symptom associated with or caused by cellproliferation or a cellular hyperproliferative disorder such as aneoplasia, tumour or cancer, or metastasis. It may lead to improvedsurvival. A satisfactory clinical endpoint of a treatment method inaccordance with the invention is achieved, for example, when there is anincremental or a partial reduction in severity, duration or frequency ofone or more associated pathologies, adverse symptoms or complications,or inhibition or reversal of one or more of the physiological,biochemical or cellular manifestations or characteristics of cellproliferation or a cellular hyperproliferative disorder such as aneoplasia, tumour or cancer, or metastasis. A therapeutic benefit orimprovement therefore may be, but is not limited to destruction oftarget proliferating cells (e.g., neoplasia, tumour or cancer, ormetastasis) or ablation of one or more, most or all pathologies, adversesymptoms or complications associated with or caused by cellproliferation or the cellular hyperproliferative disorder such as aneoplasia, tumour or cancer, or metastasis. However, a therapeuticbenefit or improvement need not be a cure or complete destruction of alltarget proliferating cells (e.g., neoplasia, tumour or cancer, ormetastasis) or ablation of all pathologies, adverse symptoms orcomplications associated with or caused by cell proliferation or thecellular hyperproliferative disorder such as a neoplasia, tumour orcancer, or metastasis. For example, partial destruction of a tumour orcancer cell mass, or a stabilization of the tumour or cancer mass, sizeor cell numbers by inhibiting progression or worsening of the tumour orcancer, can reduce mortality and prolong lifespan even if only for a fewdays, weeks or months, even though a portion or the bulk of the tumouror cancer mass, size or cells remain.

Specific non-limiting examples of therapeutic benefit include areduction in neoplasia, tumour or cancer, or metastasis volume (size orcell mass) or numbers of cells, inhibiting or preventing an increase inneoplasia, tumour or cancer volume (e.g., stabilizing), slowing orinhibiting neoplasia, tumour or cancer progression, worsening ormetastasis, or inhibiting neoplasia, tumour or cancer proliferation,growth or metastasis.

In an embodiment of the invention, administration of the checkpointinhibitor, in combination therapy with an immunomodulator, preferably awhole cell, heat-killed Mycobacterium, provides a detectable ormeasurable improvement or overall response according to the irRC (asderived from time-point response assessments and based on tumourburden), including one of more of the following: (i) irCR—completedisappearance of all lesions, whether measurable or not, and no newlesions (confirmation by a repeat, consecutive assessment no less than 4weeks from the date first documented), (ii) irPR—decrease in tumourburden ≥50% relative to baseline (confirmed by a consecutive assessmentat least 4 weeks after first documentation).

An invention method may not take effect immediately. For example,treatment may be followed by an increase in the neoplasia, tumour orcancer cell numbers or mass, but over time eventual stabilization orreduction in tumour cell mass, size or numbers of cells in a givensubject may subsequently occur.

Additional adverse symptoms and complications associated with neoplasia,tumour, cancer and metastasis that can be inhibited, reduced, decreased,delayed or prevented include, for example, nausea, lack of appetite,lethargy, pain and discomfort. Thus, a partial or complete decrease orreduction in the severity, duration or frequency of an adverse symptomor complication associated with or caused by a cellularhyperproliferative disorder, an improvement in the subjects quality oflife and/or well-being, such as increased energy, appetite,psychological well-being, are all particular non-limiting examples oftherapeutic benefit.

A therapeutic benefit or improvement therefore can also include asubjective improvement in the quality of life of a treated subject. Inan additional embodiment, a method prolongs or extends lifespan(survival) of the subject. In a further embodiment, a method improvesthe quality of life of the subject.

In a most preferred embodiment, administration of the checkpointinhibitor, in combination therapy with an immunomodulator, preferably awhole cell, heat-killed Mycobacterium results in a clinically relevantimprovement in one or more markers of disease status and progressionselected from one or more of the following: (i): overall survival, (ii):progression-free survival, (iii): overall response rate, (iv): reductionin metastatic disease, (v): circulating levels of tumour antigens suchas carbohydrate antigen 19.9 (CA 19.9) and carcinoembryonic antigen(CEA) or others depending on tumour, (vii) nutritional status (weight,appetite, serum albumin), (viii): pain control or analgesic use, (ix):CRP/albumin ratio.

Treatment with heat-killed whole cell M. vaccae and M. obuense givesrise to more complex immunity including not only the development ofinnate immunity and type-1 immunity, but also immunoregulation whichmore efficiently restores appropriate immune functions.

The immunomodulator according to the invention is administered incombination with a checkpoint inhibitor.

In a preferred embodiment, the checkpoint inhibition therapy comprisesadministration of a blocking agent, selected from a cell, protein,peptide, antibody or antigen binding fragment thereof, directed againstCTLA-4, PD-1, PD-L1, TIM-3, BTLA, VISTA, LAG-3 and combinations thereof.

In another preferred embodiment, the checkpoint inhibition therapycomprises administration of a sub-therapeutic amount and/or duration ofa blocking agent, selected from a cell, protein, peptide, antibody orantigen binding fragment thereof, directed against CTLA-4, PD-1, PD-L1,TIM-3, BTLA, VISTA, LAG-3 and combinations thereof.

In a further preferred embodiment, the checkpoint inhibition therapycomprises administration of a blocking agent, selected from a cell,protein, peptide, antibody or antigen binding fragment thereof, directedagainst PD-1 and/or, PD-L1.

In a further preferred embodiment, the checkpoint inhibition therapycomprises administration of a blocking agent, selected from a cell,protein, peptide, antibody or antigen binding fragment thereof, directedagainst PD-1 and/or, PD-L1, simultaneously, separately or sequentiallywith administration of a blocking antibody or antigen binding fragmentthereof, directed against CTLA-4.

The term “combination” as used throughout the specification, is meant toencompass the administration of the checkpoint inhibitor simultaneously,separately or sequentially with administration of the Mycobacterium.Accordingly, the checkpoint inhibitor and the Mycobacterium may bepresent in the same or separate pharmaceutical formulations, andadministered at the same time or at different times.

Thus, a Mycobacterium and the checkpoint inhibitor may be provided asseparate medicaments for administration at the same time or at differenttimes.

Preferably, a Mycobacterium and checkpoint inhibitor are provided asseparate medicaments for administration at different times. Whenadministered separately and at different times, either the Mycobacteriumor checkpoint inhibitor may be administered first; however, it issuitable to administer checkpoint inhibitor followed by theMycobacterium. In addition, both can be administered on the same day orat different days, and they can be administered using the same scheduleor at different schedules during the treatment cycle.

In an embodiment of the invention, a treatment cycle consists of theadministration of a Mycobacterium daily, weekly fortnightly or monthly,simultaneously with checkpoint inhibitor weekly. Alternatively, theMycobacterium is administered before and/or after the administration ofthe checkpoint inhibitor.

In another embodiment of the invention, the Mycobacterium isadministered to the patient before and after administration of acheckpoint inhibitor. That is, in one embodiment, the immunomodulator isadministered to the patient before and after checkpoint inhibitor.

Dose delays and/or dose reductions and schedule adjustments areperformed as needed depending on individual patient tolerance totreatments.

Alternatively, the administration of checkpoint inhibitor may beperformed simultaneously with the administration of the effectiveamounts of the Mycobacterium.

The immunomodulator, preferably a whole cell heat-killed Mycobacterium,may be administered to the patient via the parenteral, oral, sublingual,nasal or pulmonary route. In a preferred embodiment, the immunomodulatoris administered via a parenteral route selected from subcutaneous,intradermal, subdermal, intraperitoneal, intravenous and intravesicularinjection.

More preferably, administration by the parenteral route does notcomprise intratumoural injection of mycobacterial cell wall extract.

The subject whom is to undergo checkpoint inhibition therapy accordingto the present invention may do so simultaneously, separately orsequentially with administration of the immunomodulator, preferably awhole cell heat-killed Mycobacterium.

In an aspect of the invention, the effective amount of theimmunomodulator may be administered as a single dose. Alternatively, theeffective amount of the immunomodulator may be administered in multiple(repeat) doses, for example two or more, three or more, four or more,five or more, ten or more, or twenty or more repeat doses. Theimmunomodulator may be administered between about 4 weeks and about 1day prior to checkpoint inhibition therapy, such as between about 4weeks and 1 week, or about between 3 weeks and 1 week, or about between3 weeks and 2 weeks. Administration may be presented in single ormultiple doses.

In a preferred embodiment of the invention there is a Mycobacterium foruse in the treatment of neoplastic disease in combination with acheckpoint inhibitor wherein said Mycobacterium is administered to thesubject before, concurrently with and/or after the checkpoint inhibitoris administered.

In another preferred embodiment of the invention is a method oftreating, reducing, inhibiting or controlling a neoplasia, tumour orcancer in a subject, wherein said method comprises simultaneously,separately or sequentially administering to the subject, (i) acheckpoint inhibitor, and (ii) an immunomodulator, preferably a wholecell heat-killed Mycobacterium, wherein said method results in enhancedtherapeutic efficacy relative to administration of the checkpointinhibitor or immunomodulator alone.

In another preferred embodiment of the invention is a method oftreating, reducing, inhibiting or controlling a neoplasia, tumour orcancer in a subject, wherein said method comprises simultaneously,separately or sequentially administering to the subject, (i) acheckpoint inhibitor, and (ii) an immunomodulator, preferably a wholecell heat-killed Mycobacterium, wherein said checkpoint inhibitiontherapy comprises administration of a blocking agent, selected from acell, protein, peptide, antibody or antigen binding fragment thereof,directed against CTLA-4, PD-1, PD-L1, TIM-3, BTLA, VISTA, LAG-3 andcombinations thereof.

In another preferred embodiment of the invention is a method oftreating, reducing, inhibiting or controlling a neoplasia, tumour orcancer in a subject, wherein said method comprises simultaneously,separately or sequentially administering to the subject, (i) acheckpoint inhibitor, and (ii) an immunomodulator, preferably a wholecell heat-killed Mycobacterium, wherein said checkpoint inhibitiontherapy comprises administration of a blocking agent, selected from acell, protein, peptide, antibody or antigen binding fragment thereof,directed against PD-1 and/or PD-L1.

In yet another preferred embodiment of the invention is a method oftreating, reducing, inhibiting or controlling a neoplasia, tumour orcancer in a subject, wherein said method comprises simultaneously,separately or sequentially administering to the subject, (i) acheckpoint inhibitor, and (ii) an immunomodulator, preferably a wholecell heat-killed Mycobacterium, wherein said checkpoint inhibitiontherapy comprises administration of a blocking agent, selected from acell, protein, peptide, antibody or antigen binding fragment thereof,directed against CTLA-4, PD-1, PD-L1, TIM-3, BTLA, VISTA, LAG-3 andcombinations thereof, wherein said checkpoint inhibition therapycomprises administration of a sub-therapeutic amount and/or duration ofsaid blocking antibody or antigen binding fragment thereof.

In yet another embodiment of the invention is a method of treating,reducing, inhibiting or controlling a neoplasia, tumour or cancer in asubject, wherein said method comprises simultaneously, separately orsequentially administering to the subject, (i) two or more checkpointinhibitors, and (ii) an immunomodulator, preferably a whole cellheat-killed Mycobacterium, wherein said checkpoint inhibition therapycomprises administration of a blocking agent, selected from a cell,protein, peptide, antibody or antigen binding fragment thereof, directedagainst CTLA-4, PD-1, PD-L1, TIM-3, BTLA, VISTA, LAG-3 and combinationsthereof, wherein said checkpoint inhibition therapy optionally comprisesadministration of a sub-therapeutic amount and/or duration of saidblocking agent, selected from a cell, protein, peptide, antibody orantigen binding fragment thereof.

In one embodiment of the present invention, the Mycobacterium may be inthe form of a medicament administered to the patient in a dosage form.

A container according to the invention in certain instances, may be avial, an ampoule, a syringe, capsule, tablet or a tube. In some cases,the mycobacteria may be lyophilized and formulated for resuspensionprior to administration.

However, in other cases, the mycobacteria are suspended in a volume of apharmaceutically acceptable liquid. In some of the most preferredembodiments there is provided a container comprising a single unit doseof mycobacteria suspended in pharmaceutically acceptable carrier whereinthe unit dose comprises about 1×10⁶ to about 1×10¹⁰ organisms. In somevery specific embodiments the liquid comprising suspended mycobacteriais provided in a volume of between about 0.1 ml and 10 ml, or betweenabout 0.3 ml and 2 ml or between about 0.5 ml and 2 ml. The foregoingcompositions provide ideal units for immunotherapeutic applicationsdescribed herein.

Embodiments discussed in the context of a methods and/or composition ofthe invention may be employed with respect to any other method orcomposition described herein. Thus, an embodiment pertaining to onemethod or composition may be applied to other methods and compositionsof the invention as well.

In some cases non-pathogenic heat-killed mycobacteria are administeredto specific sites on or in a subject. For example, the mycobacterialcompositions according to the invention, such as those comprising M.obuense in particular, may be administered adjacent to tumours oradjacent to lymph nodes, such as those that drain tissue surrounding atumour. Thus, in certain instances sites administration of mycobacterialcomposition may be near the posterior cervical, tonsillar, axillary,inguinal, anterior cervical, sub-mandibular, sub mental orsuperclavicular lymph nodes.

The immunomodulator may be administered for the length of time thecancer or tumour(s) is present in a patient or until such time thecancer has regressed or stabilized. The immunomodulator may also becontinued to be administered to the patients once the cancer or tumourhas regressed or stabilised.

Mycobacterial compositions according to the invention will comprise aneffective amount of mycobacteria typically dispersed in apharmaceutically acceptable carrier. The phrases “pharmaceutically orpharmacologically acceptable” refers to molecular entities andcompositions that do not produce an adverse, allergic or other untowardreaction when administered to an animal, such as, for example, a human,as appropriate. The preparation of an pharmaceutical composition thatcontains mycobacteria will be known to those of skill in the art inlight of the present disclosure, as exemplified by Remington'sPharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, Moreover,for animal (e.g., human) administration, it will be understood thatpreparations should meet sterility, pyrogenicity, general safety andpurity standards. A specific example of a pharmacologically acceptablecarrier as described herein is borate buffer or sterile saline solution(0.9% NaCl).

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives {e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavouring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329).

In a preferred embodiment, the immunomodulator is administered via aparenteral route selected from subcutaneous, intradermal, subdermal,intraperitoneal, intravenous and intravesicular injection. Intradermalinjection enables delivery of an entire proportion of the mycobacterialcomposition to a layer of the dermis that is accessible to immunesurveillance and thus capable of electing an anti-cancer immune responseand promoting immune cell proliferation at local lymph nodes.

Though in highly preferred embodiments of the invention mycobacterialcompositions are administered by direct intradermal injection, it isalso contemplated that other methods of administration may be used insome case. Thus in certain instances heat-killed mycobacteria of thepresent invention can be administered by injection, infusion, continuousinfusion, intravenously, intradermally, intraarterially,intraperitoneally, intralesionally, intravitreally, intravaginally,intrarectally, topically, intratumourally, intramuscularly,intraperitoneally, subcutaneously, subconjunctival, intravesicularlly,mucosally, intrapericardially, intraumbilically, intraocularally,orally, intracranially, intraarticularly, intraprostaticaly,intrapleurally, intratracheally, intranasally, topically, locally,inhalation (e.g. aerosol inhalation), via a catheter, via a lavage, orby other method or any combination of the forgoing as would be known toone of ordinary skill in the art (see, for example, Remington'sPharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990). Morepreferably, administration by the parenteral route does not compriseintratumoural injection of mycobacterial cell wall extract.

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the present invention will be apparentto those skilled in the art without departing from the scope and spiritof the present invention. Although the present invention has beendescribed in connection with specific preferred embodiments, it shouldbe understood that the invention as claimed should not be unduly limitedto such specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in biochemistry and immunology or related fields areintended to be within the scope of the following claims.

The invention is further described with reference to the followingnon-limiting Example.

Example 1

Adult C57BL/6 mice were injected subcutaneously on the flank with 10⁵cells from a pancreatic cancer cell line obtained from KPC mice(Hingorani et al. Cancer Cell, 2005, 7:469-48). These murine pancreaticcancer cells bear mutations in Kras, p53 and Pdx-Cre (Hingorani et al.Cancer Cell, 2005, 7:469-48).

When the injected tumour cells had grown to become a palpable tumour(day 0), mice were left untreated or received treatment with:

-   -   1) 0.1 mg M. obuense NTCT 13365/mouse, subcutaneously        alternating injections in the scruff of the neck with those at        the base of the tail on alternating days over 5 day period with        a 2 day break for the length of the study;    -   2) 10 mg/kg anti-PD-L1 mAb intraperitoneally once weekly;    -   3) the combination of anti-PD-L1 and M. obuense NTCT 13365 at a        dose and schedule described above for the two compounds used        singly.

Tumour growth was monitored over the course of the study to determinewhether the treatment administered had an effect on reducing tumour sizeand improving prospects of survival.

Data presented in FIG. 1 show that mice which received the treatmentcombination of anti-PD-L1 and M. obuense NTCT 13365 demonstrated acontinued reduction in tumour size and appeared to control the tumour.This reduction in tumour size was more pronounced compared to micereceiving either treatment alone. Mice left untreated had uncontrolledtumour growth and soon succumbed to the disease.

1-18. (canceled)
 19. In a method of treating, reducing, inhibiting orcontrolling a sarcoma in a human patient by more than one intravenousadministration of a therapeutically effective amount of a checkpointinhibitor selected from the group consisting of an anti-PD-1 antibody, ahuman or humanized anti-PD-L1 antibody, and an anti-CTLA-4 antibody tothe patient, the improvement comprising: further administering to thehuman patient two or more doses of whole cell, heat-killed Mycobacteriumobuense, wherein 0.01 mg to 1 mg of the whole cell, heat-killedMycobacterium obuense is administered to the human patient per dose,wherein the whole cell, heat-killed Mycobacterium obuense isadministered simultaneously, separately or sequentially with respect tothe checkpoint inhibitor, with each of the whole cell, heat-killedMycobacterium obuense and checkpoint inhibitor being administered onmultiple days, and wherein the method results in enhanced therapeuticefficacy relative to administration of the checkpoint inhibitor alone.20. The method of claim 19, wherein the amount of the whole cell,heat-killed Mycobacterium obuense administered is from 10⁷ to 10⁹ cellsper dose.
 21. The method of claim 19, wherein the two or more doses ofthe whole cell, heat-killed Mycobacterium obuense comprise three or moredoses of the whole cell, heat-killed Mycobacterium obuense.
 22. Themethod of claim 21, wherein the three or more doses of the whole cell,heat-killed Mycobacterium obuense are administered over multiple weeks.23. The method of claim 19, wherein the amount of the whole cell,heat-killed Mycobacterium obuense administered is from 0.1 to 1 mg perdose.
 24. The method of claim 19, wherein the improvement comprisesadministering the whole cell, heat-killed Mycobacterium obuense adjacentto the sarcoma in the human patient.
 25. The method of claim 19, whereinthe whole cell, heat-killed Mycobacterium obuense is administered beforeadministration of the checkpoint inhibitor.
 26. The method of claim 19,wherein the whole cell, heat-killed Mycobacterium obuense is a roughvariant.
 27. The method of claim 19, wherein the sarcoma is metastatic.28. The method of claim 19, wherein enhanced therapeutic efficacy ismeasured by increased overall survival time, or increasedprogression-free survival.
 29. The method of claim 19, wherein enhancedtherapeutic efficacy is measured by a decrease or stabilization ofsarcoma tumor size.
 30. The method of claim 19, wherein enhancedtherapeutic efficacy is measured by increased quality of life.
 31. Themethod of claim 19, wherein the checkpoint inhibitor is an anti-PD-1antibody.
 32. The method of claim 19, wherein the checkpoint inhibitoris a human or humanized anti-PD-L1 antibody.
 33. The method of claim 19,wherein the checkpoint inhibitor is an anti-CTLA-4 antibody.
 34. Themethod of claim 19, wherein the checkpoint inhibitor is administered asa combination of human or humanized anti-PD-1 and/or anti-PD-L1 and/oranti-CTLA-4 antibodies.
 35. The method of claim 19, wherein the sarcomais a primary neoplasia.
 36. The method of claim 19, wherein the sarcomais a soft tissue sarcoma.
 37. The method of claim 19, wherein the M.obuense is administered via the parental, oral, sublingual, nasal orpulmonary route.
 38. The method of claim 19, wherein the M. obuense isadministered via a parental route selected from subcutaneous,intradermal, subdermal, intraperitoneal, or intravenous.